| Rabies virus(RABV)is a non-segmented negative-stranded RNA virus of the Rhabdoviridae and induces a globa public health threat and fatal viral zoonosis.Members of these viral genome approximately 12 kb,and the genome encodes five structure protein in the order of nucleoprotein(N),phosphoprotein(P),matrix protein(M),glycoprotein(G),and RNA-dependent RNA polymerase(RdRp;also termed large protein(L).This zoonotic virus spread to human via biting or scratching of RABV infected all terrestrial mammals such as bats,dogs and skunks.Unlike other infectious disease,as usual,human rabies vaccine immunized as post-exposure prophylaxis(PEP)not as pre-exposure.Furthermore,appropriate usages of rabies vaccine and human rabies immunoglobulin(HRIG)have proven highly effective in the prevention of infections and death.However,there are still more than 59000 human died for rabies every single year.More than 95% human deaths are reported in the developing countries in Asia and Africa,which most probably caused by the limited control of the infectous animals and the relative expensive vaccines in these countries.With the development of modern cell culture techniques,viral vaccine productions in quality,purity,efficiency and safety have been made much progressed,the economical costs are also obviously decreased.Give those,a better cell adaption and passage stable RABV remains to be explored,and the specific nucleotide sites that proceed cell adaption need further to be investigated.In the present study,two strains of RABVs named as CNIM1701 and CNIM1702 were isolated from seven rapid cattle.The whole genome sequencing of the two RABVs were sequenced and submitted to NCBI and obtained genebank numbers as KY649620 and MF172976.Through the phylogenetic analysis,these two RABVs belongs to the wild type RABVs of Mongolian and Russia stain over the world.To explore the difference of nucleotide site between wild type RABVs and attenuated RABVs,the CNIM1701 strain of RABV was performed a serial passage in sucking mice brains.After 8 generation in mice brain passage,the CNIM1701 was weakly adapted to NA and BSR cells.Moreover,there are 8 mutation of amino acids in between the G and L after passed in mice brain,such as G protein of 170 amino acids by serine(ser)S into alanine(gla)A,367 amino acids from proline(pro)P to serine(ser)S and proves that the mutation is a gradual process.Through evolutionary tree analysis and gene mutation site comariesion,G367 and G83 are two key amino acid sites to distinguish the cell adaptive and wild type RABVs.Finally,we used infections cDNA clones of SAD to rescue single or double amino acids swapping RABVs at positions G367 and G83,and the pathogenicity of these virus will be done in the next step.In a word,G367 and G83 are the key amino acids site determined the RABVs cell adaption and pathogenicity,which would help the development of high-yieldRABVs vaccine. |
